Biochem. J. (2011) 435, 553562 (Printed in Great Britain) doi:10.

1042/BJ20102121
553
REVIEW ARTICLE
Regulation of spermiogenesis, spermiation and bloodtestis barrier
dynamics: novel insights from studies on Eps8 and Arp3
C. Yan CHENG
1
and Dolores D. MRUK
The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10065, U.S.A.
Spermiogenesis in the mammalian testis is the most critical post-
meiotic developmental event occurring during spermatogenesis in
which haploid spermatids undergo extensive cellular, molecular
and morphological changes to form spermatozoa. Spermatozoa
are then released fromthe seminiferous epitheliumat spermiation.
At the same time, the BTB (bloodtestis barrier) undergoes
restructuring to facilitate the transit of preleptotene spermatocytes
from the basal to the apical compartment. Thus meiotic divisions
take place behind the BTB in the apical compartment to form
spermatids. These germ cells enter spermiogenesis to transform
into elongating spermatids and then into spermatozoa to replace
those that were released in the previous cycle. However, the mole-
cular regulators that control spermiogenesis, in particular the
dynamic changes that occur at the Sertoli cellspermatid interface
and at the BTB, are not entirely known. This is largely due to
the lack of suitable animal models which can be used to study
these events. During the course of our investigation to develop
adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide]
as a potential male contraceptive, this drug was shown to
accelerate spermiation by inducing the release of premature
spermatids from the epithelium. Using this model, we have
identied several molecules that are crucial in regulating the
actin lament network and the unique adhesion protein complex
at the Sertoli cellspermatid interface known as the apical ES
(ectoplasmic specialization). In the present review, we critically
evaluate these and other ndings in the literature as they
relate to the restricted temporal and spatial expression of two
actin regulatory proteins, namely Eps8 (epidermal growth factor
receptor pathway substrate 8) and Arp3 (actin-related protein 3),
which regulate these events.
Key words: adjudin, bloodtestis barrier, intermediate lament,
seminiferous epithelial cycle, spermatogenesis, tubulobulbar
complex.
INTRODUCTION
Most changes in cell morphology, plasticity and movement
resulting from cues received from the environment, growth and
development, stress, cytokines, toxicants or during pathogenesis
are regulated by the actin-, intermediate lament- and/or
tubulin-based cytoskeletons [16]. This applies to virtually all
epithelial cells, including those in the seminiferous epithelium
of the mammalian testis. Interestingly, the actin network in the
seminiferous epithelium, which is composed of only Sertoli cells
and germ cells which are at different stages of development (i.e.
spermatogonial stemcells, spermatogonia, primary and secondary
spermatocytes, spermatids and spermatozoa) (Figure 1), is notably
different from actin networks found in other epithelia in several
ways. First, actin lament bundles found in Sertoli cells at
the ES {ectoplasmic specialization; a testis-specic atypical
AJ (adherens junction) found at the Sertoli cellspermatid and
SertoliSertoli cell interface known as the apical and basal ES
respectively [79]} (Figures 1 and 2) are non-contractile [9,10],
even though motor proteins (e.g. myosin VIIa) are present at
the ES [10,11]. Secondly, actin lament bundles are tightly
packed, arranged parallel to the Sertoli cell plasma membrane, and
sandwiched in between apposing cell membranes of Sertoli cell
spermatid or SertoliSertoli cell and cisternae of endoplasmic
reticulum (Figures 1 and 2). Although other studies have shown
that the ES is one of the strongest adhesive junctions [12,13], it
still is subjected to extensive restructuring. This thus facilitates the
transit of spermatids across the epitheliumduring spermiogenesis,
as well as the transit of preleptotene spermatocytes across the
BTB (bloodtestis barrier) at stage VIII of the seminiferous
epithelial cycle of spermatogenesis [14,15]. Thirdly, although
Sertoli cells cultured in vitro are highly motile and capable
of traversing the membranes of transwell (i.e. bicameral) units
similar to metastatic cancer cells [16,17], Sertoli cells are in
fact not motile in vivo. Instead, they are static, nurse-like
cells needed for germ cell development with each Sertoli cell
engulng approx. 3050 developing germ cells, but they do
alter their cell shape to acommodate morphological changes of
spermatids during spermiogenesis. Moreover, Sertoli cells are
the only structural and scaffolding cells in the seminiferous
epithelium that confer BTB function via co-existing TJs (tight
junctions), basal ES, desmosomes and gap junctions located near
the basement membrane in the seminiferous epithelium, since
microvessels in the interstitial space between tubules contribute
relatively little to the BTB. On a nal note, all of these junctions
link to either the actin, the intermediate lament or the tubulin
network, and they are interconnected structurally and functionally.
In the present review, we critically discuss results from recent
studies relating to two actin regulatory proteins: Eps8 (epidermal
growth factor receptor pathway substrate 8) (Figure 3) and Arp3
Abbreviations used: adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide; AJ, adherens junction; Arp, actin-related protein; ARPC, Arp2/3
complex subunit; BTB, bloodtestis barrier; Cdc42, cell division cycle 42; Eps8, epidermal growth factor receptor pathway substrate 8; ES, ectoplasmic
specialization; F-actin, lamentous actin; PAR, partitioning defective protein; N-WASP, neuronal WASP; SCAR/WAVE, suppressor of cAMP receptor/WASP
family verprolin homologous; TJ, tight junction; WASP, WiskottAldrich syndrome protein.
1
To whom correspondence should be addressed (email Y-Cheng@popcbr.rockefeller.edu).
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Figure 1 A schematic drawing illustrating the relative location of apical ES, basal ES at the BTB, gap junction and desmosome-like junction in the
seminiferous epithelium of adult mammalian testes
It is noted that the BTB is constituted by coexisting TJ, apical ES, desmosome-like junction and gap junction (see text). Apical ES and basal ES are an actin-based testis-specic AJ type restricted to
the Sertoli cellspermatid (step 819 spermatid) interface and SertoliSertoli interface respectively, typied by the actin lament bundles sandwiched in between cisternae of ES and the apposing
plasma membranes of Sertolispermatid or SertoliSertoli cell (see Figure 2). Gap junction (actin-based cellcell junction) and desmosome-like junction (intermediate lament-based cellcell
junction) are found at the SertoliSertoli, Sertolispermatogonium, Sertolispermatocyte and Sertolipre-step 8 spermatid interface. Integrated membrane proteins at the ES, gap junction, TJ and
desmosome-like junction are linked to the actin network (for ES, gap junction and TJ) or intermediate laments (for desmosome-like junction and hemidesmosome) via adaptors [31,84,85].
(actin-related protein 3) (Figure 4) that work together to modulate
actin dynamics within the seminiferous epithelium, in particular
at the apical ES and the BTB. Eps8 is a multifunctional actin
regulatory protein [1820]. Depending on its association with
different binding partners; IRSp53 (insulin receptor tyrosine
kinase substrate p53), Abi-1 (Abelson interacting protein-1),
or Sos1 (son of sevenless 1) and Abi-1, the Eps8 protein
complex can regulate actin bundling [21], capping of actin
barbed-ends [22] or Rac (a GTPase) activation [23] (Figure 4),
all of which regulate actin dynamics [20]. Arp3, on the other
hand, is a component of the Arp 2/3 complex, which is one of
the major powerhouses that creates a branched actin network
in cells. Although the functional Arp2/3 nucleation complex
is composed of seven subunits, Arp2, Arp3 and ARPCs
(Arp2/3 complex subunit) 15 [2426] (Figure 4), the Arp2/3
complex is not active. Instead it is activated by WASP
(WiskottAldrich syndrome protein) family proteins, namely N-
WASP (neuronal-WASP), SCAR/WAVE (suppressor of cAMP
receptor/WASP family verprolin homologous) and cortactin to
initiate actin nucleation/branching on a pre-existing actin lament
[20,25,27,28] (Figure 4). It is worth noting that besides Eps8
and Arp3, many other proteins that work with Eps8 and the
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Figure 2 Anatomical features of the ES in the testis
(AC) Corresponding cross-sections of seminiferous tubules at stages VI, VIII and XIV of the seminiferous epithelial cycle in the adult rat testis, illustrating the intimate relationship between germcells
at different stages of their development with Sertoli cells in the seminiferous epithelium. (A) Developing step 18 spermatids in a stage VI tubule are embedded deep within the seminiferous epithelium
and anchored to Sertoli cells by the apical ES (see circled area) only. The BTB created by coexisting TJs, basal ES, desmosome-like and gap junctions of adjacent Sertoli cells near the basement
membrane (see boxed area) divides the epithelium into the apical and the basal compartments, which remains closed at this stage. However, step 19 spermatids aligned at the adluminal edge of the
tubule lumen in preparation for spermiation in a stage VIII tubule (B) when the BTB undergoes extensive restructuring (open) to facilitate the transit of preleptotene spermatocytes from the basal to
the apical compartment. Thereafter, all step 19 spermatids transformed to sperm are released into the tubule lumen at late stage VIII and round spermatids transform to step 14 spermatids at stage XIV
shown in (C), and late spermatocytes undergo meiosis at stage XIV of the epithelial cycle (C) to give rise to spermatids. Spermatocytes undergoing meiosis are clearly visible and marked with a red
asterisk illustrating late anaphase. Es, elongating spermatid; Rs, round spermatid; Sg, spermatogonium; Pm, peritubular myoid cell; SC, Sertoli cell. (D) The apical ES shown in the circled region in
(AC) is magnied in this electron micrograph, illustrating the ultrastructural features of an apical ES, which is typied by the presence of actin lament bundles (white arrowheads) sandwiched
in between cisternae of endoplasmic reticulum (ER) and the apposing plasma membrane of the Sertoli cell (green arrowhead) and the elongating spermatid (red arrowhead), and these typical
ultrastructural features are restricted only to the Sertoli cell side at the apical ES. Chromatin is condensed and packed into the spermatid head as the nucleus. The developing acrosome (Ac) capping
part of the nucleus is also visible. (E) The basal ES at the BTB as shown in the boxed region in (AC) is magnied in this electron micrograph. The basal ES is ultrastructurally identical with the
apical ES except that its typical feature, namely the actin lament bundles (black arrowheads) that are sandwiched in between cisternae of ER and the apposing plasma membranes of two adjacent
Sertoli cells (apposing green arrowheads) are found within both Sertoli cells. The basal ES coexists with TJs as illustrated by yellow arrowheads illustrating the kisses between apposing Sertoli cell
plasma membranes. It is noted that the BTB is found near the basement membrane. Scale bars in (AC), (D) and (E) are 20 m, 0.2 m and 0.5 m respectively.
Arp2/3 complex have recently been identied in the testis [29,30].
In the present review, we will focus our discussion largely
on the actin-based cytoskeleton and the signicance of actin
regulatory proteins in spermiogenesis and spermiation, as well
as BTB dynamics since few studies are found in the literature
relating to the roles of the intermediate lament- or tubulin-based
cytoskeletons in spermatogenesis [20,31].
THE SEMINIFEROUS EPITHELIAL CYCLE AND SPERMIOGENESIS
In the mammalian testis, spermiogenesis begins immediately after
meiosis II, which takes place in a specialized microenvironment
in the apical compartment of the seminiferous epithelium at stage
XIV, XII or VI of the seminiferous epithelial cycle in the rat,
mouse or human respectively (for reviews, see [32,33]). During
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Figure 3 Different effects of Eps8 on the actin-based cytoskeleton network in the seminiferous epithelium of the rat testis
Depending on its various binding partners, Eps8 can modulate the actin network via its interaction with different proteins. Abi-1, Abelson interacting protein-1; IRSp53, insulin receptor tyrosine
kinase substrate p53; Sos1, son of sevenless 1.
Figure 4 Differential effects of Eps8 and the Arp2/3 protein complex on the actin-based cytoskeleton network
(A) Eps8 functions to stabilize actin lament bundles found at the apical and basal ES in the seminiferous epithelium. (B) The Arp2/3 protein complex is composed of 7 subunit proteins, namely Arp2,
Arp3 and ARPC15, which must be activated by N-WASP and SCAR/WAVE N-WASP. This protein complex can induce nucleation (branching) of an existing actin lament, thereby de-stabilizing the
apical and basal ES, which is necessary for the movement of developing spermatids and the transit of preleptotene spermatocytes across the BTB respectively.
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spermiogenesis, round spermatids (whose precursor cells are
the secondary spermatocytes) undergo extensive morphological,
cellular and molecular transformations via a series of 19, 16
or 6 steps in the rat, mouse or human respectively. This is
typied by the condensation of genetic material which forms
the nucleus in the spermatid head, the development of the acro-
some partly covering the nucleus and the elongation of the tail
along with the formation of tightly packed mitochondria in the
midpiece (for reviews, see [3336]) (Figure 2). Interestingly,
development of the acrosome which caps the spermatid nucleus
during spermiogenesis has allowed investigators to classify
developing spermatids by the PAS (periodateSchiff) reaction and
to divide the seminiferous epithelium into discrete stages (IXIV,
IXII or IVI in the rat, mouse or human respectively). In essence,
each stage is comprised of unique cellular associations between
developing germ cells (in particular spermatids) and Sertoli cells,
and these cellular associations can be clearly observed in cross-
sections of seminiferous tubules [37,38] (Figure 2). Once fully
developed elongated spermatids have formed and residual bodies
have been phagocytosed by Sertoli cells which occurs at the end of
spermiogenesis, spermatozoa are released from the seminiferous
epithelium (i.e. spermiation) into the tubule lumen so that they
can be transported to the epididymis for further maturation (for
reviews, see [33,39]).
Besides the highly organized and intricate cellular events that
occur during spermiogenesis (for reviews, see [11,33,36,40,41]),
the transformation of spermatids is also marked by other
important cellular events which are critical for the completion
of spermatogenesis. First, CT (cancer/testis) antigens and germ
cell-specic antigens (for reviews, see [7,4244]), many of which
are expressed only transiently during post-meiotic germ cell
development throughout spermiogenesis, are sequestered away
from the systemic circulation by the BTB. The BTB is formed by
1517 days post-partum in the rat [45] and at puberty in humans
by 1213 years of age [46], which also plays a signicant role
in mammals to confer, at least in part, an immune-privilege
status to the testis [47]. Thus the BTB prohibits the production
of anti-sperm auto-antibodies, at least in post-meiotic spermatids
during spermiogenesis and spermiation, which would otherwise
induce male infertility. Secondly, there is extensive junction
restructuring at the Sertoli cellspermatid interface to allow the
transit of developing spermatids across the epitheliumuntil stages
VIIVIII when elongated spermatids properly line-up near the
luminal edge in preparation for spermiation (for reviews, see
[15,20,4850]). As such, the events of germ cell movement are
synchronized precisely with the events of germ cell development.
In the following sections, we highlight several important studies
that have reported interactions among different actin regulatory
proteins, as well as discuss their highly restricted spatial and
temporal expression patterns in the seminiferous epithelium. We
also discuss how actin regulatory proteins interact with polarized
protein complexes at the Sertoli cellspermatid interface (i.e.
apical ES) to induce changes in cell plasticity and polarity,
thereby facilitating rapid transformations in cell shape via
protein endocytosis, recycling, transcytosis and endosome- or
ubiquitin-mediated degradation.
THE ACTIN NETWORK AT THE ECTOPLASMIC SPECIALIZATION
During spermiogenesis, a unique AJ type known as the apical
ES emerges at the interface between Sertoli cells and elongating
spermatids at step 8 and beyond in the rat (Figures 1 and 2)
(for reviews, see [8,11,14]). Once it appears, it becomes the only
adhesive device that anchors spermatids to Sertoli cells, replacing
desmosome-like and gap junctions that are present between
Sertoli cells and pre-step 8 spermatids or between Sertoli cells
spermatocytes. Besides being the only anchoring device used by
developing spermatids, the apical ES is also known to maintain
proper spermatid polarity and orientation within the epithelium
so that the heads of all elongating/elongated spermatids point
towards the basement membrane (for reviews, see [8,10,48]). This
permits for tight arrangement of a maximal number of spermatids
during spermatogenesis (Figure 2). Although this apical ES
function has been largely a speculation for decades [51,52], it
was only recently reported that this polarity function is made
possible by the presence of several polarity protein complexes
at the apical ES, namely the PAR3 (partitioning defective
protein 3)/PAR6/Cdc42 (cell division cycle 42), the PALS1
(protein-associated with Lin-Seven 1)/PATJ (PALS1-associated
TJ protein)/CRB (Crumbs) and the Scribble/LGL1/2 (lethal giant
larvae 1/2) protein complexes (for reviews, see [14,48,53]).
Although these polarity proteins were initially identied in
Caenorhabditis elegans, a homologue for each of these proteins
has been found in mammals and reported to be restricted to
the TJ to confer cell polarity [48,54,55]. Interestingly, many
of these proteins have been demonstrated to be components of
the apical ES, and their roles in conferring spermatid polarity
have been demonstrated [56,57].
The most obvious and unique ultrastructural feature of the
apical ES is the precise arrangement of actin lament bundles that
are sandwiched in between apposing plasma membranes of the
Sertoli cell/elongating spermatid and the cisternae of endoplasmic
reticulum (Figures 1 and 2). The apical ES is limited only to the
Sertoli cell without any notable ultrastructural features visible in
the spermatid (Figure 2) (for a review, see [14]). The ES is also
found at the SertoliSertoli cell interface (i.e. basal ES) and re-
stricted to the BTB near the basement membrane, coexisting with
TJs, desmosomes and gap junctions. However, unlike the apical
ES, the ultrastructural features of the basal ES are found within
both Sertoli cells (Figure 2) (for a review, see [14]). Thus the basal
ES is believed to work with TJs at the BTBto confer adhesion and
cell polarity to Sertoli cells in the testis so that Sertoli cell nuclei
and some of their organelles (e.g. Golgi apparatus, endoplasmic
reticulum) can maintain their polarized localization. This is
different from other epithelia in which TJs exclusively play a
signicant role to confer cell polarity (for reviews, see [48,54,55]).
Moreover, the apical ESat the Sertoli cellspermatid (step 8 and
beyond) interface was shown to be a signicantly stronger testis-
specic AJ compared with desmosome gap junctions at the Sertoli
cellspermatid (pre-step 8) or the Sertoli cellspermatocyte
interface when the adhesive force between these junctions
was quantied by using a micropipette pressure transducing
system [12]. Interestingly, although the adhesive force that was
required to detach post-step 8 spermatids from Sertoli cells
(i.e. disrupting the apical ES, 8.8210
7
pN) was almost twice
as much as that needed to detach pre-step 8 spermatids from
Sertoli cells (i.e. desmosome gap junctions, 4.7310
7
pN)
[12], the apical ES undergoes extensive restructuring from step
8 to 19 spermatids to coincide with changes in shape and
location which result from their morphological transformation
and movement across the seminiferous epithelium during the
epithelial cycle of spermatogenesis (Figures 1 and 2). Studies
have shown that this rapid restructuring of the apical ES during
spermatogenesis is mediated by the differential effects of the
actin bundling/barbed end capping protein Esp8 [21,22,58] and
the actin nucleation/branching Arp2/3N-WASPCdc42 protein
complex [26,5962] via their highly regulated temporal and
spatial expression patterns [29,30]. This is discussed in the next
section, and a hypothetical model based on these ndings is
depicted in Figure 5.
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Figure 5 A hypothetical model based on the highly restricted temporal and spatial expression of Eps8 and Arp3 at the apical and basal ES in the seminiferous
epithelium, which affects the status of actin lament bundles at these sites to facilitate germ cell transit
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Molecular mechanisms of spermiogenesis 559
AN EMERGING CONCEPT OF ACTIN REGULATION WHICH MEDIATES
APICAL ES RESTRUCTURING TO FACILITATE SPERMATID
MOVEMENT ACROSS THE SEMINIFEROUS EPITHELIUM DURING
SPERMIOGENESIS
Recent studies have shown the expression of Eps8 and Arp3
to be stage-specic during the seminiferous epithelial cycle of
spermatogenesis, and these proteins localized predominantly at
the apical and basal ES [29,30]. For instance, it was shown that
Eps8, an actin-bundling protein, localized largely to the apical
ES at stages VVII of the epithelial cycle, co-localizing with
F-actin (lamentous actin) and coinciding with the time when
actin lament bundles were needed to maintain the integrity
of the apical ES [30]. However, at stage VIII, just prior to
spermiation, the apical ES degenerates via internalization of
adhesion proteins to give rise to an ultrastructure visible by
uorescence or electron microscopy as the tubulobulbar complex
[39,63]. The tubulobulbar complex seems to resemble a giant
clathrin-based endocytic vesicle [39,63] and is typied by
invaginations of Sertoli cell membranous structures [63,64], and
during this time the presence of Eps8 diminished to a virtually
undetectable level when examined by immunohistochemistry or
dual-labelled immunouorescence analysis [30]. On the other
hand, Arp3, an actin nucleation/branching protein, also localized
predominantly at the apical ES at stage VVII, co-localizing
with F-actin as well, and likewise its level became virtually
undetectable at stage VIII when tubulobulbar complexes were
present at the Sertoli cellelongated spermatid interface [29].
These ndings coupled with studies using inhibitors or specic
siRNA (small interfering RNA) duplexes which blocked the
function of Arp3 [29] or Eps8 [30] respectively, have prompted
us to hypothesize that their unique and highly restricted temporal
and spatial expression/localization patterns at the apical ES at
stages VVII of the epithelial cycle facilitate the rapid de-
bundling, bundling and branching of actin laments to allow the
transit of elongating/elongated spermatids across the seminiferous
epithelium during spermiogenesis (Figure 5). However, during
spermiation, when there is virtually no apical ES restructuring,
the expression of Eps8 and Arp3 is barely detectable (e.g. at early
stage VIII just prior to spermiation). It is possible that during this
time, components of the endocytosed adhesion protein complexes
can be recycled via transcytosis to the newly developed
elongating spermatids via spermiogenesis for the assembly of the
new apical ES (Figure 5). In short, this novel mechanism allows
rapid restructuring of the apical ES and efcient utilization of
apical ES proteins via transcytosis and recycling (Figure 5).
The model depicted in Figure 5 is further supported by
studies using an in vivo model involving the administration of
adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide].
When adult rats are treated with adjudin, disruption of the
apical ES was preferentially induced prior to the disruption of
desmosome-like and gap junctions between Sertoli cells and pre-
step 8 spermatids or spermatocytes [65]. Subsequent in vitro
studies using the micropipette pressure transducing systemindeed
conrmed that the apical ES is more susceptible to adjudin
treatment compared with desmosome-like junctions at the Sertoli
cell/germ cell interface since it took signicantly less force to
pull post-step 8 spermatids apart from Sertoli cells compared
with pre-step 8 spermatids following treatment of Sertoli/germ
cell cocultures with adjudin [13]. A signicant loss of Eps8 at the
apical ESwas detected within 11 h in rats treated with adjudin, and
by 24 h virtually no immunoreactive Eps8 was found at the apical
ES[30]. Although Arp3 did not diminish signicantly at the apical
ES by 24 h post-treatment, immunoreactive Arp3 at the apical ES
became disorganized and mis-localized, moving fromthe concave
to the convex side of the spermatid head, possibly to induce
premature actin branching before spermiation [29]. Collectively,
these ndings illustrate that adjudin induces actin branching at
the apical ES via changes in the localization of Apr3. This is
concomitant with the de-bundling of actin laments at the apical
ES because a loss of Eps8 at this site induced spermatid release,
in a sense mimicking the normal physiological spermiation that
takes place at late stage VIII of the epithelial cycle, except that it
occurred prematurely in response to adjudin treatment (Figure 5).
Nevertheless, additional research is needed to further conrm
the hypothesis depicted in Figure 5. For instance, it remains to be
determined biochemically whether protein endocytosis, recycling
and transcytosis indeed occur at the apical ES, analogous to
proteins at the basal ES and TJ at the BTB [66,67].
ACTIN DYNAMICS AT THE BTB
In virtually all cell epithelia, with the notable exception of
the seminiferous epithelium, TJs (zonula occludens) reside
at the apical region of cells, followed by AJs (zonula
adherens) and desmosomes (macula adherens), which collectively
create the junctional complex (for reviews, see [68,69]). Gap
junctions usually lie behind the junctional complex, and then
hemidesmosomes and focal contacts are found at the cellmatrix
interface [68]. It is because of this morphological intimacy
between TJs and AJs that a damage to the former, such as that
induced by xenobiotics or toxicants, can lead to a disruption of the
latter, and vice versa, and an eventual dissolution of the junctional
complex (for reviews, see [68,69]). However, in the testis, a well-
dened junctional complex does not even exist since TJs lie closest
to the basement membrane (a modied form of the extracellular
matrix [70]), and they coexist with AJs and desmosomes, as
well as with gap junctions to constitute the BTB, such that each
In (A), elongating spermatids are anchored to the Sertoli cell in the seminiferous epithelium via apical ES as shown in the left-hand panel. Integral membrane proteins at the apical ES are contributed
mostly by the integrinlaminin (e.g. 61-integrinlaminin 33 3) complex, nectinafadin and N-cadherin-catenin [8]. It is worth noting that these apical ES proteins are actin-based adhesion
protein complexes. Intracellularly, however, the intracellular tail of cadherin can associate with the Armadillo family protein plakoglobin (also known as -catenin) and plakophilins, which bind to the
desmosomal cytoskeletal adaptor protein desmoplakin [20,86]. Thus N-cadherin can be seen to co-localize with intermediate laments by uorescence microscopy [87]. The integrity of the apical
ES is maintained by Eps8 with the proper orientation of the actin lament bundles. This actin network becomes disorganized, which is mediated by Arp2/3 protein complex during spermiogenesis
to facilitate the movement of elongating spermatid across the epithelium (middle panel), and at stage VIII of the epithelial cycle, actin lament bundles are disrupted entirely to facilitate the release
of spermatozoa at spermiation (left-hand panel). These changes are also facilitated by endocytosis of integral membrane proteins at the apical ES so that the internalized endocytic vesicles can be
transcytosed and recycled to assemble the new apical ES in recently formed step 8 spermatids during spermiogenesis. The loss of integral membrane proteins at the apical ES via endocytosis thus
further destabilizes the apical ES. In (B), left-hand panel, an intact BTB is illustrated, which is maintained by the restricted expression of Eps8 needed for the integrity of actin lament bundles. At
the time of BTB restructuring at stage VIII of the epithelial cycle, the expression of Eps8 diminishes. This is replaced by the Arp2/3 protein complex, which destabilizes actin lament bundles via
the formation of a branched actin network, facilitating the internalization of integral membrane proteins [e.g. occludin, connexin 43 (Cx43)] (right-hand panel). This event of protein endocytosis is
aided by an increase in the phosphorylation of integral membrane proteins induced by activated FAK (focal adhesion kinase), activated c-Src and/or p38 MAPK (mitogen-activated protein kinase)
[88,89]. Previous studies have also illustrated the involvement of polarity proteins: PAR6, PAR3 and 14-3-3 (also known as PAR5) and Cdc42, in endocytic vesicle-mediated protein trafcking events
(e.g. protein endocytosis and recycling) at the Sertoli cell BTB [56,57,90]. Endocytosed proteins can be targeted for endosome-mediated degradation or be recycled and transcytosed to another site,
further destabilizing the BTB. ZO-1 (zonula occludens-1) and PKP-2 (plakophilin-2) are the corresponding adaptor proteins of the TJ protein occludin and the gap junction (GJ) protein Cx43.
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junctional type (e.g. TJ) is not segregated from the others (e.g.
basal ES) (for reviews, see [8,11,14]) (Figure 2). The BTB also
physically divides the seminiferous epithelium into the basal and
the apical compartment with post-meiotic germcell development,
namely spermiogenesis and spermiation, taking place in the apical
compartment behind the BTB (Figure 1). BTB integrity, however,
cannot be compromised, even transiently, during spermiogenesis
to avoid the production of anti-sperm antibodies, since numerous
specic autoantigens arise in spermatids during spermiogenesis
and spermiation, so that the BTB confers immune privilege to the
testis [47].
During spermiogenesis, extensive restructuring of the apical
ES occurs as discussed above. Thus why doesnt restructuring
of the apical ES during the transit of spermatids across the epi-
thelium induce disruption to the BTB? Recent studies on the
highly restricted temporal and spatial expression of Eps8 and
Arp2 have shed light on a novel mechanism (Figure 5) [29,30].
For instance, the expression of Eps8 at the BTBis high at all stages
of the epithelial cycle to maintain tightly packed actin lament
bundles that constitute basal ES function (Figure 5), except at
stage VIII when the BTB undergoes extensive restructuring to
facilitate the transit of preleptotene spermatocytes [30]. This is
also the stage when Eps8 expression at the BTB considerably
diminishes [30] to induce de-bundling of actin laments, which
facilitates breakdown of the basal ES and the BTB. Interestingly,
the expression of Arp3 is also induced considerably at the BTB
at stage VIII of the epithelial cycle. Thus this allows actin
branching, increasing the uidity and plasticity of the BTB.
This concomitant decline in Eps8 [30] and surge in Arp3 at
the BTB [29] at stage VIII of the epithelial cycle contributes
to a state where the basal ES is disrupted by reducing the
rigid actin network at the BTB. This then facilitates the transit
of preleptotene spermatocytes at the site (Figure 5). Due to
the tightly regulated temporal and spatial expression of these
two actin regulators, apical or basal ES restructuring that takes
place in the apical compartment or BTB respectively, will not
provoke a disruption of the other junction, in a way segregating
restructuring at opposite ends of the Sertoli cell epitheliumduring
the seminiferous epithelial cycle of spermatogenesis (Figure 5).
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
In the present review, we have proposed a novel mechanism
that is based on the restricted temporal and spatial expression of
two actin regulators, Eps 8 (an actin-bundling-inducing protein)
and Arp3 (an actin nucleation/branching inducing protein)
(Figure 5). Changes in the plasticity of the actin lament
network at the apical and basal ES as a result of actin
lament bundling/debundling and actin branching allow timely
disruption of the ES throughout the seminiferous epithelial cycle.
This also allows the segregation of restructuring events that occur
at the apical and basal ES so that spermatid movement during
spermiogenesis does not compromise the integrity of the BTB.
However, much more work is needed to ne-tune the hypothesis
depicted in Figure 5. For instance, what are the downstream
and upstream signalling events involving Eps8 or Arp3? What
are the roles of other actin regulatory proteins, such as nebulin
[71], WASP [72], motor proteins (e.g. myosin) [1], GTPases
(e.g. Cdc42) [1,48], formins [25,26,73,74], prolin [7577], the
colincoroninAip1 protein complex [78] and phospholipids
[79] in Eps8- and Arp3-mediated events or other pertinent events
in the seminiferous epithelium [26,59]? How do actin dynamics
contribute to the events of protein endocytosis, recycling and
transcytosis that occur during the epithelial cycle in particular at
the apical ES during spermiogenesis? Do they work similarly as
found in other epithelia [8083]? Some of these questions should
be carefully tackled by functional experiments in the years to
come.
FUNDING
Studies from this laboratory were supported by grants from the National Institutes of
Health [grant numbers NICHD R01 HD056034 and R01 HD056034-02S1 (to C.Y.C.); U54
HD029990 Project 5 (to C.Y.C.); and R03 HD061401 (to D.D.M.)].
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Received 20 December 2010/31 January 2011; accepted 3 February 2011
Published on the Internet 13 April 2011, doi:10.1042/BJ20102121
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2011 Biochemical Society